Recurrence and metastatic dissemination of breast cancers account for a significant morbidity and mortality in women, and effective means of treating this subset of patients remains elusive. Our long-term goal is to develope an effective and safe gene therapy for invasive breast cancer. The objective of this proposal is to determine the therapeutic efficacy and safety of intravenously (IV)- administered, genetically-modified endothelial cells (GMEC) co-expressing interleukin-2 (IL-2) and herpes simplex virus thymidine kinase (HSV-TK) transgenes in metastatic breast cancer-bearing mice. Our preliminary studies show that:(a) IV-administered, IL-2-expressing endothelial cells selectively migrate into sites of pulmonary metastases of breast cancer, express the recombinant IL-2 at the local tumor sites, and induce partial regression of tumor metastases, and ( b) HSV- TK transgene-expressing endothelial cells induce apoptotic death of HSV-TK-negative endothelial or breast cancer cells in co- culture after exposure to ganciclovir (GCV). Based upon these findings, we hypothesize that following IV administration, GMEC expressing a combination of IL-2 and HSV-TK transgenes can target sites of breast tumors. These cells will then respond to tumor- derived angiogenic factors by proliferation, incorporation into the developing capillary network, and expression of transgenes that will induce an effective anti-tumor response. This study is to test our hypothesis. The Specific Aims of this proposal are: Aim number 1: To construct bicistronic retroviral vectors containing HSV-TK and/or E. coli lacZ genes with intervening IRES fragments, and generate endothelial cells stably expressing HSV-TK and/or lacZ transgenes (LacZ/HSV-TK GMEC). Aim number 2: To determine a) the efficiency of IV-injected, LacZ/HSV-TK GMEC targeting of metastatic sites of breast tumor, and b) the effects of HSV-TK gene expression with GCV treatment upon tumor growth and animal survival. Aim number 3: To determine whether the administration of a mixture of IL-2 and LacZ/HSV-TK GMEC followed by GCV treatment can induce an effective, specific, and long-term anti-tumor immune response, and abrogate lung metastases of a breast tumor in mice. The proposed studies should provide the framework for the eventual design of a clinical trial of this therapeutic strategy.